PROJECT SUMMARY Type 2 diabetes mellitus (t2DM) predisposes patients to debilitating cardiovascular disorders, including acute ischemia/reperfusion (I/R) events and chronic myocardial infarction (MI), both of which promote sudden cardiac death. Many of the pathophysiological complications of t2DM can be linked to hyperglycemia-mediated mitochondrial ROS overproduction that is secondary to mitochondrial division (fission). Of importance to mitochondrial fission is the dynamin related protein 1 (DRP1), a member of the conserved dynamin GTPase superfamily. The functional importance of DRP1 is underscored by the fact that its over-expression promotes mitochondrial membrane potential depolarization and cell death whereas its silencing or chemical inhibition attenuates these processes. The implications of altered DRP1 and mitochondrial fission for modulating myocardial function and arrhythmias in t2DM remain unknown and will be examined in the context of acute I/R injury and chronic MI. The activity of DRP1 is regulated by several kinases and phosphatases including the AMP- related kinase (AMPK), a master metabolic sensor that is central in the pathophysiology of t2DM. By controlling the balance between ATP generating and consuming processes, AMPK also regulates mitochondrial function in the settings of I/R Injury and diabetic cardiomyopathy. The overall premise of this project is based on the following lines of evidence: 1) DRP1 controls mitochondrial division in various cell types, including myocytes; 2) Decreased mitochondrial fission protects against reactive oxygen species (ROS)-induced mitochondrial depolarization, mPTP opening, and apoptosis; 3) DRP1-related mitochondrial fission is required for hyperglycemia-mediated ROS overproduction in various cell types; 4) Acute ROS overproduction in I/R promotes electrical dysfunction and arrhythmia by destabilizing the mitochondrial membrane potential through the regenerative process of mitochondrial ROS-induced ROS-release; 5) Chronic ROS overproduction promotes adverse structural and mechanical remodeling; and 6) In diabetes, AMPK activation inhibits mitochondrial fission by altering DRP1 phosphorylation at specific serine residues in endothelial cells. The central tenant of this proposal is that mitochondrial fission and its regulation by an AMPK-DRP1 axis plays a central role in t2DM-related cardiac dysfunction and arrhythmia. In Aim 1, we will determine the role of DRP1-mediated mitochondrial fission and its regulation by AMPK in the susceptibility of the diabetic heart to acute ROS-related reperfusion arrhythmias. In Aim 2, we will determine the extent to which impairment in the AMPK-DRP1 axis contributes to post-MI structural and electro-mechanical remodeling and arrhythmia susceptibility in the diabetic heart. In Aim 3, we will test the therapeutic efficacy of targeting DRP1-mediated mitochondrial fission in reversing post-MI cardiac dysfunction and arrhythmia propensity in the diabetic heart. Completion of these studies will yield new mechanistic insights into the role and regulation of mitochondrial fission in t2DM, and uncover novel mitochondria-targeted therapeutic approaches for this major public health epidemic.